The primary objective of this research proposal is to elucidate the mechanism of the NAD-Malic enzyme from the parasitic nematode, Ascaris suum. NAD-Malic enzyme is a member of a class of pyridine-nucleotide- dependent enzymes that catalyze the oxidative decarboxylation of beta- hydroxy acids. Based on homology amongst t different enzymes in the class of pyridine-linked oxidative decarboxylases, the metal-independent phosphogluconate dehydrogenase (6-PGDH) is in a structural class different from that of the metal-dependent isocitrate (ICDH) and isopropylmalate (IMPDH) dehydrogenases, and both differ from the malic enzyme, which also requires a divalent metal ion activator. Three dimensional structures have been solved for 6-PGDH an ICDH, but no structure is yet available for a malic enzyme. Much is presently known of the mechanism of male enzyme, including information on the nature of the transition states for oxidation and decarboxylation. There still interesting questions, however, regarding the coupling of oxidation and decarboxylation, and the relationship between enzyme structure and catalysis for the malic enzyme subclass. In the present application, it is propose to continue studies on the mechanism of the malic enzyme. The overall goal will be achieved via the following Specific Aims. Mechanistic Studies via Kinetic Isotope Effects. A number of questions remain concerning transition state structure and the nature of coupling oxidation and decarboxylation. Multiple primary deuterium/beta-secondary tritium isotope effects with label at C-3 of malate will be used to distinguish between stepwise and concerted oxidative decarboxylation as the dinucleotide substrate is changed. alpha-Secondary tritium isotope effects with tritium at the 4 position of the nicotinamide ring of NAD will be measured to obtain information on the extent of transfer of the hydride in the transition state for oxidation. To determine whether hydrogen tunneling and coupled motion occurs, the D/T method of Saunders/Klinman will be used to determine primary and secondary tritium effects. Structure-Activity via Site-Directed Mutagenesis. Site-directed mutagenesis will be used to probe the metal, malate and NAD binding sites, the identity of the general acid an general base catalytic groups, residues lining the reaction coordinate for the hydride transfer reaction, and an residues required for setting up the active, catalytic conformation of the enzyme. Structure of the NAD-Malic Enzyme. The three dimensional structure of the Ascaris NAD-Malic enzyme will be obtained in collaboration with Dr. Larry DeLucas at the University of Alabama at Birmingham. In addition, spectral probe such as fluorescence, and CD will be used with NAD, and 3-APAD bound in the absence and presence of Mg2 and malate. In addition, trials are already underway to crystallize the E:3- APAD complex. Very seldom doe one observe true transition state changes in enzymatic reactions. In the case of the malic enzyme, changes either have or may have been induced in the coupling of two of the chemical steps, and there is evidence of an increase in hydrogen tunneling with change in metal ion activator.